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[Buddy]

Hello,
I am trying to do a genome shuffling experiemnt that requires me to form yeast protoplasts (before a fusion step).

I have looked at several protocols online and am planning on using a papain to break cell walls in hypotonic solution. I have tried this protocol and am not sure how successful protoplast formation was.

By light microscopy, yeast cells before and after enzyme treatment and after appear to be relatively round, perhaps "puffed up" as one might expect from the hypertonic treatment.

Even after long incubation times, the cells seem intact(by micrscopy, again), which makes me believe that the enzyme is not very active.

How can I tell if the rounded cells, post enzyme treatment, are true protoplasts?

Thanks,
Buddy